Notch signaling is essential during the early stages of T cell development within the thymus. Notch is also constitutively active in many T acute lymphoblastic leukemias. However, the molecular mechanisms by which Notch influences T cell development and T cell leukemias are incompletely understood. Early T lineage progenitors (ETPs) are the most primitive T cell progenitors that have been identified in the mouse thymus, and possess lineage plasticity. We have recently shown that the majority of ETPs, in fact, retain myeloid lineage potential at the single cell level. This myeloid potential is most clearly evident when Notch signals are withdrawn, and preliminary evidence suggests that the canonical Notch target Hes1 is involved in Notch-mediated repression of the myeloid potential of ETPs. We, therefore, propose to investigate the mechanism by which Hes1 constrains the myeloid lineage potential of ETPs. As this application is for proposed salary support for only one year, will address this goal through the following single aim: Aim1. To evaluate the mechanism by which the canonical Notch target Hes1 suppresses the myeloid lineage potential of ETP. In this aim, we will assess the ability of myeloid-committed progenitors to differentiate into mature myeloid cells in the presence of over-expressed Hes1. In addition, we will analyze the ability of ETPs to divert towards the myeloid fate when Hes1 is absent. We will also determine if Hes1- mediated suppression of the myeloid fate is through direct repression of C/EBPalpha and/or PU.1, key meyloid transcriptional regulators. Notch receptor signals are critical for T cell development and mutations associated with Notch have been found in a significant fraction of T acute lymphoblastic leukemias. However, the way by which Notch influences uncommitted progenitors to become T cells is unclear. Through this proposal, we aim to better understand the role that Notch plays in funneling progenitors down the T cell pathway by investigating the manner in which one of its known targets, Hes1, blocks at least one of the alternative lineage potentials that the earliest T cell progenitors can adopt.